Saliva is the principle protective agent for the mouth and thus is of primary importance to oral health maintenance. Perturbations of salivary secretory mechanisms can consequently lead to serious oral health problems. The objective of this project is to study the membrane and cellular processes that underlie the phenomenon of salivary fluid secretion and thus to contribute to our understanding of the fluid secretory process. Because similar secretory mechanisms are thought to be common to a number of other tissues, this information should be of rather broad applicability and interest. During the present reporting period we have continued our in-depth studies of the salivary Na-K-2Cl cotransporter (NKCC1). We have also extended some of our methodology to examine the structural properties of the water channel AQP1 and the calcium ion channel Trp1. NKCC1 is thought to be the major Cl entry pathway into salivary acinar cells and thus to be primarily responsible for driving Cl secretion, and thereby fluid secretion, in salivary glands. Obtaining a better understanding of this protein and its behavior in acinar cells will improve our knowledge of salivary function and dysfunction, as well as possibly providing indications of how to treat the latter. Over the past year we have concentrated on two projects involving NKCC1: (i) Identification and characterization of the functional regions of NKCC1. In order to identify and characterize the functional regions of NKCC1 we are carrying out cysteine scanning mutagenesis. The principle of this method is to replace amino acids at or close to possible functional residues with cysteine and then to evaluate the effects of reacting the mutated protein with sulfhydryl reagents. Since highly specific sulfhydryl reagents with a wide range of physical properties are available, it is possible to obtain considerable information about the environment of reacting sites. One of these mutants (A483C) has proven to be particularly interesting since it renders the protein sensitive to inhibition by several sulfhydryl reagents that have no effect on the wild type transporter. This residue is located in membrane spanning region 6, a highly conserved portion of the protein that our previous studies indicate is critical for function. Inhibition of NKCC1 at this site is independent of the presence of sodium and potassium but requires chloride, indicating that this effect is sensitive to the conformation of the protein. Mutations of surrounding amino acids indicate that the sensitive region corresponds to one face of an alpha helix suggesting that this is the secondary structure of membrane spanning segment 6. (ii) Identification of the sites responsible for NKCC1 dimerization. In past studies we have established that NKCC1 exists as a homodimer in the plasma membrane. In order to identify the regions of the protein responsible for dimer formation we have expressed a series of truncation mutants of NKCC1 in HEK293 cells. These experiments indicate that mutants containing 6 or more membrane spanning segments of NKCC1 tend to form dimers, however, the most significant contributor to dimerization appears to be the cytosolic C terminus of the protein. The involvement of the C terminus in dimer formation is currently being investigated using the yeast two hybrid system as well as continued studies with truncation mutants. In last years annual report we described the development of a new system for determining the transmembrane topology of an integral membrane protein using intact cells. Briefly stated we construct a fusion protein consisting of the green fluorescent protein followed by a portion of the putative membrane spanning region of the protein then a glycosylation tag. By assaying for glycosylation of the tag we can establish whether it is located in the lumen of the endoplasmic reticulum or the cytosol. Thus by incrementally increasing the length of the membrane spanning region of the protein included in the construct we can trace the topology of the protein as it crosses back and forth across the membrane. We have used this system to explore the membrane integration of the water channel AQP1. Previous studies employing in vitro translation indicated that some membrane spanning segments of AQP1 were not initially integrated into the membrane and that a post-translational conformational change was necessary for their integration, resulting in the final conformation of the protein. In contrast, our results with intact cells indicate that AQP1 integrates into the membrane cotranslationally, suggesting that the isolated microsomal system does not always mimic results obtained with intact cellular machinery. We are now using this same system to study the topology of the calcium channel Trp1 which may be involved secretory signaling in salivary and other exocrine glands. The current model of the topology of this protein is based on homology and hydrophobicity analyses; no previous direct topological studies have been carried out. Our results differ in several ways from this current model. Most dramatically our data indicate that the "pore" region of the channel is much more deeply embedded in the membrane than previously suspected and suggest that the pore is actually a part of complexly folded hydrophobic pocket. Studies attempting to delineate the involvement of the neighboring sixth membrane spanning segment of Trp1 in this pocket are now underway.